ABSTRACT Articular cartilage is avascular tissue and cannot naturally heal. Thus, there is an unmet clinical need to effectively treat partial thickness or full thickness osteochondral lesions arising from traumatic joint injuries or conditions such as osteochondritis dissecans. If not successfully treated, focal lesions will eventually progress to post-traumatic osteoarthritis, a debilitating joint disease causing substantial pain at tremendous social and economic cost. There is growing interest in the field to design strategies to generate an integrated osteochondral (OC) tissue consisting of articular cartilage transitioning to bone tissue as a means to treat partial and full thickness cartilage lesions. Producing such tissue from a single cell population would constitute a major advancement in the field. The goal of this study is to generate native-like human osteochondral tissue by an innovative approach utilizing 3D woven PCL scaffolds in combination with microRNA (miRNA)-transduced cartilage progenitor cells (CPCs). The Guilak Lab has demonstrated the effectiveness of using 3D composite PCL scaffolds for engineering functional cartilage or bone tissue. Research in the McAlinden Lab has shown that miR-181a/b enhances hypertrophic chondrocyte differentiation of CPCs while miR-138 appears to suppress this terminal differentiation event, thereby favoring articular/hyaline cartilage production. We therefore hypothesize that stable osteochondral tissue can be produced by site-specific over-expression of miR-138 and miR- 181a/b in progenitor cells within 3D composite PCL scaffolds. This hypothesis will be tested in two specific aims. Specific Aim 1 will involve a step-wise approach to generate bi-layered scaffolds consisting of articular cartilage (induced by miR-138) and hypertrophic cartilage (induced by miR-181a/b), the latter of which we predict will be an efficient template for bone formation when placed in osteogenic induction medium. Specific Aim 2 will involve testing how in vitro-generated tissues within bi-layered scaffolds can be maintained in an uncontrolled in vivo environment following subcutaneous implantation in immunodeficient mice. This innovative, high-risk project provides a unique opportunity to combine expertise on biomaterials and bioengineering (Guilak Lab) with basic molecular and cell biology expertise on miRNA-mediated regulation of skeletal cell differentiation (McAlinden Lab). New knowledge will be obtained on the effectiveness of targeting specific miRNAs to guide formation of articular and hypertrophic cartilage for tissue engineering, and if native-like osteochondral tissue can be subsequently generated. This work will provide the foundation for future pre-clinical in vivo studies in larger animals to determine if in vitro-generated osteochondral tissue or lentiviral-loaded PCL scaffolds seeded with skeletal progenitor cells can be implanted into cartilage lesions as a promising treatment strategy for cartilage repair.